Various techniques have been used to count events or items contained in fluid samples. Visual counting, using magnification and highly specialized recognition software, has been used. Conductivity counting also has been used to detect bands of DNA separated via electrophoresis. A bipolar pulse method has been used for such conductivity measurements. This method uses a bipolar pulse signal and makes use of the property that the sample cell parallel capacitance is orders of magnitude smaller than that of the series double-layer capacitance. This method is described in Bipolar Pulse Technique for Fast Conductance Measurements, by D. E. Johnson and C. G. Enke, published in Analytical Chemistry, Vol. 42, No. 3, March 1970, and hereby incorporated by reference into this application.
The bipolar pulse method provides a reasonably fast and accurate method of measuring conductivity of a fluid sample cell. The problem identified and discussed in the article cited above is the existence of two distinct capacitances exhibited by a typical fluid sample cell. In a such a cell, an electrolyte buffer solution is used as the base or background material. The samples of interest are then placed in this buffer solution. A pair of electrodes are positioned at opposite sides of the sample cell, and the conductivity across the electrodes is measured. As an item of interest (i.e., an article with conductivity measurably different from that of the buffer solution) passes the electrodes, the device detects the conductivity change. Unfortunately, the existence of capacitance due to the cell itself leads to inaccuracies in the measurements.
The Johnson and Enke article notes that a fluid sample cell exhibits a capacitance in series with the resistance of the sample and a capacitance in parallel with the cell resistance. The first capacitance is identified as a double-layer capacitance (Cd), and the latter as the parallel cell capacitance (Cp). In the bipolar pulse method, square wave pulses are used with inverse polarity, and the measurement is made at the trailing end of the second pulse. This approach results in a charging/discharging process for the parallel cell capacitance (Cp), which is essentially complete by the time the measurement is taken. In this manner, the bipolar pulse method effectively eliminates the influence of the two cell capacitances, and produces conductivity measurements proportional to changes in the conductivity of the sample.
Though the bipolar pulse method is reasonable fast, it is not without its share of problems. Two pulses are required for a single measurement, and the pulses must be long enough to allow for the parallel cell capacitance (Cp) to fully charge yet short enough to prevent the double-layer capacitance (Cd) from significantly charging and limiting the current before it is sampled. Therefore, it is necessary to have some prior knowledge of these capacitor values in order to select the most appropriate pulse lengths. This method can take measurements up to every 40 μsec, based on use of 20 μsec pulses.
The bipolar pulse method is also dependant upon the geometry of the sample cell. Cd must be greater than Cp in order for the technique to work. When the sample cell is very small, this relationship between the capacitors is no longer true. It has been found that for individual cell counts, a sample tube diameter of approximately 50 microns is advantageous. This size tube is small enough to isolate individual cells without them clogging in the tube. This small sample tube size, however, does not work well with the bipolar pulse method because the two cell capacitances (Cd and Cp) are within less than one order of magnitude of each other. In addition, the currents thru the cell must be connected to the inverting input of an opamp to be converted to a voltage. Unless the opamp is in close proximity to the cell, the stray capacitance from the inverting input to ground can cause severe ringing in the bipolar pulse. Finally, the experiments in micro-channels using this technique tend to have a low signal to noise ratio, and it is difficult to measure less than 2% changes in conductance.
An improved apparatus and method, therefore, is needed for performing fast, accurate conductivity measurements of a fluid sample. This apparatus and method would be of particular utility for counting individual cells, such as cancer cells, contained in a fluid sample. A fast count rate is needed. A good signal to noise ratio is also needed to ensure that individual cells are not missed. To accomplish this result, the apparatus and method must effectively deal with the capacitive effects of the sample cell. The bipolar pulse method is one way to doing this, but it does not work well with small sample cells and small conductivity changes. A new design is needed. The present invention is such a design.